This research involves the characterization of a collection of temperature-sensitive mutants of Escherichia coli isolated after localized mutagenesis. Most of these show defects in protein synthesis both in vivo and in vitro. Our current work includes mapping studies by P1 phage transduction to localize the genes conferring temperature-sensitivity. In addition we have identified several mutants with electrophoretic mobility changes in certain ribosomal proteins. We are presently purifying these proteins to examine the nature of the sequence change. Ribosomal subunit assembly is defective in some of the mutants at elevated temperatures. We are isolating the accumulated ribosomal precursor particles in order to examine their protein and RNA composition. Ribosomal reconstitution assays are also being conducted to examine assembly of the altered subunits in vitro. Finally we are in the process of isolating additional mutants by modifications of the method of localized mutagenesis. Several different mutagens are being tried with the transducing phage population and phage growth on antibiotic resistant heat strains are being used to increase the specificity and frequency of isolation of associated ribosomal gene mutants in E. coli.